ipsogen BCR-ABL1 Mbcr Kit Handbook

March 2015 ipsogen BCR-ABL1 Mbcr Kit Handbook 24 Version 1 Quantitative in vitro diagnostics For use with Rotor-Gene Q, ABI PRISM, LightCycler, and SmartCycler instruments 670123 QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, GERMANY R2 1072507EN Sample & Assay Technologies

QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result. QIAGEN sets standards in: Purification of DNA, RNA, and proteins Nucleic acid and protein assays microrna research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit www.qiagen.com.

Contents Intended Use 5 Summary and Explanation 5 Disease monitoring 5 Principle of the Procedure 8 Materials Provided 10 Kit contents 10 Materials Required but Not Provided 11 Warnings and Precautions 12 General precautions 12 Reagent Storage and Handling 13 Procedure 14 Sample RNA preparation 14 Protocols Recommended standardized EAC reverse transcription 14 qpcr on Rotor-Gene Q MDx 5plex HRM or Rotor-Gene Q 5plex HRM instruments with 72-tube rotor 17 qpcr on ABI PRISM 7000, 7700, and 7900HT SDS, and LightCycler 480 Instrument 21 qpcr on LightCycler 1.2 and 2.0 Instruments 26 qpcr on the SmartCycler instrument 30 Interpretation of Results 33 Data analysis principle 33 Results 34 Troubleshooting guide 36 Quality Control 39 Limitations 40 Performance Characteristics 40 Nonclinical studies 40 Clinical studies 43 References 45 Symbols 47 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 3

Contact Information 47 Ordering Information 48 4 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Intended Use The ipsogen BCR-ABL1 Mbcr Kit is intended for the quantification of BCR-ABL p210 b2a2 or b3a2 transcripts in bone marrow or peripheral blood samples of acute lymphoblastic leukemia (ALL) or chronic myeloid leukemia (CML) patients previously diagnosed with a BCR-ABL Mbcr fusion gene (FG) event. The test is intended to evaluate the level of molecular response; results can be used for minimal residual disease follow-up. Summary and Explanation CML belongs to the group of myeloproliferative neoplasms and is in >90% of cases characterized by the presence of the Philadelphia chromosome (Ph CHRS). This chromosome is the product of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22), BCR (breakpoint cluster region) being located on chromosome 22 and the c-abl oncogene coming from chromosome 9. The corresponding fusion gene, BCR-ABL, is transcribed into an 8.5 kb mrna, with 2 junction variants b2a2 (40% of cases) and b3a2 (55% of cases). It encodes a chimeric protein, p210, with elevated tyrosine kinase activity. The b2a3 and b3a3 transcripts represent less than 5% of cases. A Ph chromosome can also be detected in 35% of adult ALL patients. Annual incidence of CML is approximately 1 2 per 100,000, and CML accounts for 20% of adult leukemias. It is characterized clinically by an excess of myeloid cells that differentiate and function normally. CML patients will be diagnosed in 90 95% of cases in the chronic or stable phase of the disease. Historically, within an average of 4 to 6 years, patients were entering an accelerated phase leading to blastic crisis and acute leukemia, which is always fatal. The advents of imatinib and more recently, second generation tyrosine kinase inhibitors (TKI), dramatically changed the natural course of the disease: most of the patients now remain in remission and deserve long-term follow-up and disease monitoring. Disease monitoring To date, the goal of CML therapy is to achieve 100% survival and Ph chromosome negativity. Disease monitoring is therefore an essential tool to assess treatment response and detect early relapse for each individual patient. Under TKI therapy, patients typically progress from hematologic to cytogenetic then molecular remission, corresponding to decreasing numbers of leukemic cells and BCR-ABL transcripts as detailed in the Figure 1 below. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 5

10 12 Diagnosis, pretreatment, or hematologic relapse 100 Number of leukemic cells 10 11 Complete hematologic response 10 10 10 1 Complete cytogenetic response 10 9 0.1 10 8 0.01 10 7 Major molecular response 0.001 10 6 0.0001 BCR-ABL ratio (according to the international scale) Undetectable transcript (complete molecular response) Figure 1. Adapted from reference 1. The standard method to estimate the tumor burden in CML patients is conventional cytogenetic analysis (G-banding) on bone marrow (BM) metaphases. Cytogenetic response is assessed on at least 20 marrow metaphases. The level of cytogenetic response is estimated on the percentage of Ph chromosome-positive metaphases (see Table 1, reference 2) However, this assessment depends on laboratory performances and has a low sensitivity, at 5% when 20 metaphases are analyzed. Real time quantitative polymerase chain reaction (qpcr) quantifying BCR-ABL Mbcr mrna, on peripheral blood (PB) specimens is now part of the disease monitoring techniques for CML on treatment. It is less invasive than conventional bone marrow metaphase cytogenetics, and more sensitive. Recommendations for CML disease monitoring have also been recently updated to incorporate new clinical evidence from clinical trials as well as improved disease monitoring objectives and tools. The most recent recommendations on response definition and monitoring of patients on imatinib come from the ELN experts (2). From a technical standpoint, efforts have been made by international experts to harmonize BCR-ABL Mbcr testing and reporting (3 5). Additionally, a reference 6 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

panel has been validated recently under auspices of the WHO, to allow a simple standardization of BCR-ABL quantification (6). Table 1. International recommendations for the management of CML patients (adapted from reference 2) Hematologic response Cytogenetic response Molecular response (BCR-ABL to control gene ratio according to the international scale) Definitions Complete: Platelet count <450 x 10 9 /liter White blood cell count <10 x 10 9 /liter Differential without immature granulocytes and with less than 5% basophils Non-palpable spleen Complete: Ph+ 0% Partial: Ph+ 1 35% Minor: Ph+ 36 65% Minimal: Ph+ 66 95% None: Ph+ >95% Complete indicates transcript nonquantifiable and nondetectable Major: 0.1 Monitoring Check every 2 weeks until complete response is achieved and confirmed, then 3 monthly unless otherwise required Check at least every 6 months until complete response is achieved and confirmed, then at least every 12 months Check every 3 months Mutational analysis in case of failure, suboptimal response or transcript level increase Complete hematologic response, cytogenetic response and molecular response should be confirmed on two subsequent occasions. Cytogenetic response is evaluated by morphologic cytogenetics of at least 20 marrow metaphases. Fluorescence in situ hybridization (FISH) of peripheral blood cells should be used only if marrow cells cannot be obtained. Molecular response is assessed on peripheral blood cells. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 7

Principle of the Procedure qpcr permits the accurate quantification of PCR products during the exponential phase of the PCR amplification process. Quantitative PCR data can be rapidly obtained, without post-pcr processing, by real-time detection of fluorescent signals during and/or subsequent to PCR cycling, thereby drastically reducing the risk of PCR product contamination. At present, 3 main types of qpcr techniques are available: qpcr analysis using SYBR Green I Dye, qpcr analysis using hydrolysis probes, and qpcr analysis using hybridization probes. This assay exploits the qpcr double-dye oligonucleotide hydrolysis principle. During PCR, forward and reverse primers hybridize to a specific sequence (Figure 2). A double-dye oligonucleotide is contained in the same mix. This probe, which consists of an oligonucleotide labeled with a 5' reporter dye and a downstream, 3'quencher dye, hybridizes to a target sequence within the PCR product. qpcr analysis with hydrolysis probes exploits the 5' 3' exonuclease activity of the Thermus aquaticus (Taq) DNA polymerase. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer. Figure 2. Schematic diagram of the BCR-ABL Mbcr FG transcript covered by the qpcr primers and probe set: ENF501 ENP541 ENR561. The number under the primers and probe refers to their nucleotide position in the normal gene transcript. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5' 3' exonuclease activity of the DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then 8 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

displaced from the target, and polymerization of the strand continues. The 3' end of the probe is blocked to prevent extension of the probe during PCR (Figure 3). This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and hence amplified during PCR. Because of these requirements, nonspecific amplification is not detected. Thus, the increase in fluorescence is directly proportional to the target amplification during PCR. Figure 3. Reaction principle. Total RNA is reverse-transcribed, and the generated cdna is amplified by PCR using a pair of specific primers and a specific internal double-dye probe (FAM TAMRA ). The probe binds to the amplicon during each annealing step of the PCR. When the Taq DNA polymerase extends from the primer bound to the amplicon, it displaces the 5' end of the probe, which is then degraded by the 5' 3' exonuclease activity of the Taq DNA polymerase. Cleavage continues until the remaining probe melts off the amplicon. This process releases the fluorophore and quencher into solution, spatially separating them and leading to an increase in fluorescence from the FAM and a decrease in fluorescence from the TAMRA. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 9

Materials Provided Kit contents ipsogen BCR-ABL1 Mbcr Kit (24) Catalog no. 670123 Number of reactions 24 ABL Control Gene Standard Dilution (10 3 copies/5 µl) C1-ABL 50 µl ABL Control Gene Standard Dilution (10 4 copies/5 µl) ABL Control Gene Standard Dilution (10 5 copies/5 µl) C2-ABL 50 µl C3-ABL 50 µl BCR-ABL Mbcr Fusion Gene Standard Dilution (10 1 copies/5 µl) BCR-ABL Mbcr Fusion Gene Standard Dilution (10 2 copies/5 µl) BCR-ABL Mbcr Fusion Gene Standard Dilution (10 3 copies/5 µl) BCR-ABL Mbcr Fusion Gene Standard Dilution (10 5 copies/5 µl) BCR-ABL Mbcr Fusion Gene Standard Dilution (10 6 copies/5 µl) Primers and Probe Mix ABL* F1-BCR- ABL Mbcr F2-BCR- ABL Mbcr F3-BCR- ABL Mbcr F4-BCR- ABL Mbcr F5-BCR- ABL Mbcr PPC-ABL 25x 50 µl 50 µl 50 µl 50 µl 50 µl 90 µl Primers and Probe Mix BCR-ABL Mbcr Fusion Gene PPF-Mbcr 25x 110 µl ipsogen BCR-ABL Mbcr Kit Handbook (English) 1 * Mix of specific reverse and forward primers for the ABL control gene plus a specific FAM TAMRA probe. Mix of specific reverse and forward primers for the BCR-ABL Mbcr fusion gene plus a specific FAM TAMRA probe. Note: Briefly centrifuge the standard dilutions and the primers and probe mixes before use. 10 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Materials Required but Not Provided When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. Reagents Nuclease-free PCR grade water Reagents for reverse transcription: The validated reagent is Superscript II (or Superscript) Reverse Transcriptase, includes 5x first-strand buffer, 100 mm DTT (Life Technologies, cat. no. 18064-022) RNase inhibitor: The validated reagent is RNaseOUT (Life Technologies, cat. no. 10777-019) Set of dntps, PCR grade Random hexamer MgCl 2 Buffer and Taq DNA polymerase: The validated reagents are TaqMan Universal PCR Master Mix (Master Mix PCR 2x) (Life Technologies, cat. no. 4304437) and LightCycler TaqMan Master (Master Mix PCR 5x) (Roche, cat. no. 04535286001) Consumables Nuclease-free aerosol-resistant sterile PCR pipet tips with hydrophobic filters 0.5 ml or 0.2 ml RNase- and DNase-free PCR tubes Ice Equipment Microliter pipet* dedicated for PCR (1 10 µl; 10 100 µl; 100 1000 µl) Benchtop centrifuge* with rotor for 0.2 ml/0.5 ml reaction tubes (capable of attaining 10,000 rpm) Real-time PCR instrument:* Rotor-Gene Q MDx 5plex HRM or other Rotor-Gene instrument; LightCycler 1.2, 2.0, or 480; ABI PRISM 7000, 7700, or 7900HT SDS; or SmartCycler instrument; and associated specific material Thermal cycler* or water bath* (reverse transcription step) * Ensure that instruments have been checked and calibrated according to the manufacturer s recommendations. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 11

Complementary reagents ipsogen BCR-ABL1 Mbcr Controls Kit (cat. no. 670191), consisting of cell lines with negative, high, and low positive expression of the BCR-ABL Mbcr fusion gene for the qualitative validation of the RNA extraction and the reverse transcription Warnings and Precautions For in vitro diagnostic use When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit and kit component. Discard sample and assay waste according to your local safety regulations. General precautions qpcr tests require good laboratory practices, including equipment maintenance, that are dedicated to molecular biology and compliant with applicable regulations and relevant standards. This kit is intended for in vitro diagnostic use. Reagents and instructions supplied in this kit have been validated for optimal performance. Further dilution of the reagents or alteration of incubation times and temperatures may result in erroneous or discordant data. PPC and PPF reagents may be altered if exposed to light. All reagents are formulated specifically for use with this test. For optimal performance of the test, no substitutions should be made. Determining transcript levels using qpcr requires both the reverse transcription of the mrna and the amplification of the generated cdna by PCR. Therefore, the entire assay procedure must be performed under RNase-/DNase-free conditions. Use extreme caution to prevent: RNase/DNase contamination, which might cause degradation of the template mrna and the generated cdna mrna or PCR carryover contamination resulting in false positive signal We therefore recommend the following. Use nuclease-free labware (e.g., pipets, pipet tips, reaction vials) and wear gloves when performing the assay. Use fresh aerosol-resistant pipet tips for all pipetting steps to avoid crosscontamination of the samples and reagents. 12 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Prepare pre-pcr master mix with dedicated material (pipets, tips, etc.) in a dedicated area where no DNA matrixes (cdna, DNA, plasmid) are introduced. Add template in a separate zone (preferably in a separate room) with specific material (pipets, tips, etc.). Handle the standard dilutions (C1 3 and F1 5) in a separate room. Reagent Storage and Handling The kits are shipped on dry ice and must be stored at 30 C to 15 C upon receipt. Minimize exposure to light of the primers and probe mixes (PPC and PPF tubes). Gently mix and centrifuge the tubes before opening. Store all kit components in original containers. These storage conditions apply to both opened and unopened components. Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results. Expiration dates for each reagent are indicated on the individual component labels. Under correct storage conditions, the product will maintain performance until the expiration date printed on the label. There are no obvious signs to indicate instability of this product. However, positive and negative controls should be run simultaneously with unknown specimens. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 13

Procedure Sample RNA preparation RNA preparation from patient samples (blood or bone marrow) must have been performed with a validated procedure. The quality of the assay is largely dependent on the quality of input RNA. We therefore recommend qualifying the purified RNA by agarose* gel electrophoresis or by using Agilent Bioanalyzer prior to analysis. Protocol: Recommended standardized EAC reverse transcription Things to do before starting Prepare dntps, 10 mm each. Store at 20 C in aliquots. Procedure 1. Thaw all necessary components and place them on ice. 2. Incubate 1 µg of RNA (1 4 µl) for 10 minutes at 70 C and immediately cool on ice for 5 minutes. 3. Centrifuge briefly (approximately 10 seconds, 10,000 rpm, to collect the liquid in the bottom of the tube). Then keep on ice. 4. Prepare the following RT mix according to the number of samples being processed (Table 2). * When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. 14 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Table 2. Preparation of RT mix Component First-Strand Buffer (supplied with Superscript II Reverse Transcriptase), 5x Volume per sample (µl) Final concentration 4.0 1x MgCl 2 (50 mm) 2.0 5 mm dntps (10 mm each, to be prepared previously and stored at 20 C in aliquots) DTT (100 mm, supplied with Superscript II Reverse Transcriptase) 2.0 1 mm 2.0 10 mm RNase inhibitor (40 U/µl) 0.5 1 U/µl Random hexamer (100 µm) 5.0 25 µm Superscript II or Superscript Reverse Transcriptase (200 U/µl) Heated RNA sample (to be added in step 5) Nuclease-free PCR grade water (to be added in step 5) 0.5 5 U/µl 1.0 4.0 50 ng/µl 0.0 3.0 Final volume 20.0 5. Pipet 16 µl of RT mix into each PCR tube. Then add 1 4 µl (1 µg) RNA (from step 3), and adjust the volume to 20 µl with nuclease-free PCR grade water (see Table 3). Table 3. Preparation of reverse transcription reaction Component Volume (µl) RT mix 16 Heated sample RNA (1 µg) 1 4 Nuclease-free PCR grade water 0 3 Final volume 20 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 15

6. Mix well and centrifuge briefly (approximately 10 seconds, 10,000 rpm, to collect the liquid in the bottom of the tube). 7. Incubate at 20 C for 10 minutes. 8. Incubate at 42 C on a thermal cycler for 45 minutes, then immediately at 99 C for 3 minutes. 9. Cool on ice (to stop the reaction) for 5 minutes. 10. Briefly centrifuge (approximately 10 seconds, 10,000 rpm, to collect the liquid in the bottom of the tube). Then keep on ice. 11. Dilute the final cdna with 30 µl of nuclease-free PCR grade water so that the final volume is 50 µl. 12. Carry out PCR according to the following protocols, according to your qpcr instrument. 16 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Protocol: qpcr on Rotor-Gene Q MDx 5plex HRM or Rotor-Gene Q 5plex HRM instruments with 72-tube rotor Using this instrument, we recommend performing all measurements in duplicate, as indicated in Table 4. Table 4. Number of reactions for Rotor-Gene Q instruments with 72-tube rotor Samples Reactions With the ABL primers and probe mix (PPC-ABL) n cdna samples ABL standard Water control n x 2 reactions 2 x 3 reactions (3 dilutions, each one tested in duplicate) 2 reactions With the BCR-ABL Mbcr primers and probe mix (PPF-Mbcr) n cdna samples Mbcr standard Water control n x 2 reactions 2 x 5 reactions (5 dilutions, each one tested in duplicate) 2 reactions Sample processing on Rotor-Gene Q instruments with 72-tube rotor We recommend testing at least 8 cdna samples in the same experiment to optimize the use of the standards and the primers and probe mixes. Each ipsogen BCR-ABL1 Mbcr Kit provides enough reagents to perform an 8-sample experiment 3 times using the 72-tube rotor. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 17

Figure 4. Suggested rotor setup for each experiment with the ipsogen BCR-ABL1 Mbcr Kit. F1 5: BCR-ABL Mbcr standards; C1 3: ABL standards; S: cdna sample; H 2 O: water control. Note: Take care to always place a sample to be tested in position 1 of the rotor. Otherwise, during the calibration step, the instrument will not perform calibration, and incorrect fluorescence data will be acquired. Fill all other positions with empty tubes. qpcr on Rotor-Gene Q instruments with 72-tube rotor Note: Perform all steps on ice. Procedure 1. Thaw all necessary components and place them on ice. 2. Prepare the following qpcr mix according to the number of samples being processed. All concentrations are for the final volume of the reaction. 18 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Table 5 describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final reaction volume of 25 µl. A pre-mix can be prepared, according to the number of reactions, using the same primer and probe mix (either PPC-ABL or PPF-Mbcr). Extra volumes are included to compensate for pipetting error. Table 5. Preparation of qpcr mix Component TaqMan Universal PCR Master Mix, 2x Primers and probe mix, 25x Nucleasefree PCR grade water Sample (to be added at step 4) Total volume 1 reaction (µl) ABL: 24+1 reactions (µl) BCR-ABL Mbcr: 28+1 reactions (µl) Final concentration 12.5 312.5 362.5 1x 1 25 29 1x 6.5 162.5 188.5 5 5 each 5 each 25 25 each 25 each 3. Dispense 20 µl of the qpcr pre-mix per tube. 4. Add 5 µl of the RT product (cdna, 100 ng RNA equivalent) obtained in the reverse transcription (see Protocol: Recommended standardized EAC reverse transcription, page 14) in the corresponding tube (total volume 25 µl). 5. Mix gently, by pipetting up and down. 6. Place the tubes in the thermal cycler according to the manufacturer recommendations. 7. Program the Rotor-Gene Q instrument with the thermal cycling program as indicated in Table 6. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 19

Table 6. Temperature profile Mode of analysis Hold Hold 2 Cycling Quantitation Temperature: 50 deg Time: 2 mins Temperature: 95 deg Time: 10 mins 50 times 95 deg for 15 secs 60 deg for 1 min with acquisition of FAM fluorescence in channel Green: Single 8. For Rotor-Gene Q instruments, select Slope Correct for the analysis. We recommend setting the threshold at 0.03. Start the thermal cycling program, as indicated in Table 6. 20 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Protocol: qpcr on ABI PRISM 7000, 7700, and 7900HT SDS, and LightCycler 480 Instrument Using 96-well-plate qpcr equipment, we recommend performing all measurements in duplicate, as indicated in Table 7. Table 7. Number of reactions using 96-well-plate qpcr equipment Samples Reactions With the ABL primers and probe mix (PPC-ABL) n cdna samples ABL standard Water control n x 2 reactions 2 x 3 reactions (3 dilutions, each one tested in duplicate) 2 reactions With the BCR-ABL Mbcr primers and probe mix (PPF-Mbcr) n cdna samples Mbcr standard Water control n x 2 reactions 2 x 5 reactions (5 dilutions, each one tested in duplicate) 2 reactions Sample processing on ABI PRISM 7000, 7700, and 7900 SDS, and LightCycler 480 Instrument We recommend testing at least 8 cdna samples in the same experiment to optimize the use of the standards and the primers and probe mixes. The plate scheme in Figure 5 shows an example of such an experiment. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 21

Figure 5. Suggested plate setup for one experiment. S: cdna sample; F1 5: BCR-ABL Mbcr standards; C1 3: ABL standards; H 2 O: water control. qpcr on ABI PRISM 7000, 7700, and 7900 SDS, and LightCycler 480 Instrument Note: Perform all steps on ice. Procedure 1. Thaw all necessary components and place them on ice. 2. Prepare the following qpcr mix according to the number of samples being processed. If using 96-well-plate qpcr equipment, we recommend performing all measurements in duplicate. All concentrations are for the final volume of the reaction. Table 8 describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final reaction volume of 25 µl. A pre-mix can be prepared, according to the number of reactions, using the same primer and probe mix (either PPC-ABL or PPF-Mbcr). Extra volumes are included to compensate for pipetting error. 22 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Table 8. Preparation of qpcr mix Component TaqMan Universal PCR Master Mix, 2x Primers and probe mix, 25x Nucleasefree PCR grade water Sample (to be added at step 4) Total volume 1 reaction (µl) ABL: 24+1 reactions (µl) BCR-ABL Mbcr: 28+1 reactions (µl) Final concentration 12.5 312.5 362.5 1x 1 25 29 1x 6.5 162.5 188.5 5 5 each 5 each 25 25 each 25 each 3. Dispense 20 µl of the qpcr pre-mix per well. 4. Add 5 µl of the RT product (cdna, 100 ng RNA equivalent) obtained in the reverse transcription (see Protocol: Recommended standardized EAC reverse transcription, page 14) in the corresponding well (total volume 25 µl). 5. Mix gently, by pipetting up and down. 6. Close the plate and briefly centrifuge (300 x g, approximately 10 seconds). 7. Place the plate in the thermal cycler according to the manufacturer recommendations. Program the thermal cycler with the thermal cycling program as indicated in Table 9 for ABI PRISM 7000, 7700, and 7900HT SDS, or Table 10 for the LightCycler 480 Instrument. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 23

Table 9. Temperature profile for ABI PRISM 7000, 7700, and 7900HT SDS Mode of analysis Standard Curve Absolute Quantitation Hold Temperature: 50 C Time: 2 minutes Hold 2 Temperature: 95 C Time: 10 minutes Cycling 50 times 95 C for 15 seconds 60 C for 1 minute with acquisition of FAM fluorescence; quencher: TAMRA Table 10. Temperature profile for LightCycler 480 Instrument Mode of analysis Detection formats Absolute Quantification ( Abs Quant ) Select Simple Probe in the Detection formats window Hold Temperature: 50 C Time: 2 minutes Hold 2 Temperature: 95 C Time: 10 minutes Cycling 50 times 95 C for 15 seconds 60 C for 1 minute with acquisition of FAM fluorescence corresponding to (483 533 nm) for LC version 01 and (465 510 nm) for LC version 02 8. For the ABI PRISM 7000, 7700, and 7900HT SDS, follow step 8a. For the LightCycler 480 Instrument, follow step 8b. 8a. ABI PRISM 7000, 7700. and 7900HT SDS: We recommend a threshold set at 0.1 as described in the EAC protocol in the analysis step on the ABI PRISM SDS and a baseline set between cycles 3 and 15. Start the cycling program, as indicated in Table 9. 24 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

8b. LightCycler 480 Instrument: We recommend a Fit point analysis mode with background at 2.0 and threshold at 2.0. Start the thermal cycling program, as indicated in Table 10. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 25

Protocol: qpcr on LightCycler 1.2 and 2.0 Instruments Using capillary instruments, we recommend measuring samples in duplicate and controls only once, as indicated in Table 11. Table 11. Number of reactions for LightCycler 1.2 and 2.0 Instruments Samples Reactions With the ABL primers and probe mix (PPC-ABL) n cdna samples ABL standard Water control n x 2 reactions 1 x 3 reactions (3 standard dilutions, each one tested once) 1 reaction With the BCR-ABL Mbcr primers and probe mix (PPF-Mbcr) n cdna samples Mbcr standard Water control n x 2 reactions 1 x 5 reactions (5 standard dilutions, each one tested once) 1 reaction Sample processing on LightCycler 1.2 and 2.0 Instruments We recommend testing at least 5 cdna samples in the same experiment to optimize the use of the standards and primers and probe mixes. The capillary scheme in Figure 6 shows an example of an experiment. 26 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Figure 6. Suggested rotor setup for each experiment with the ipsogen BCR-ABL1 Mbcr Kit. F1 5: BCR-ABL Mbcr standards; C1 3: ABL standards; S: unknown DNA sample to be analyzed; H 2 O: water control. qpcr on LightCycler 1.2 and 2.0 Instruments Note: Because of particular technological requirements, LightCycler experiments must be performed using specific reagents. We recommend to use the LightCycler TaqMan Master and to follow the manufacturer's instructions to prepare the Master Mix 5x. Note: Perform all steps on ice. Procedure 1. Thaw all necessary components and place them on ice. 2. Prepare the following qpcr mix according to the number of samples being processed. All concentrations are for the final volume of the reaction. Table 12 describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final reaction volume of 20 µl. A pre-mix can be prepared, according to the number of reactions, using the same primer and probe mix (either PPC-ABL or PPF-Mbcr). Extra volumes are included to compensate for pipetting error. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 27

Table 12. Preparation of qpcr mix Component 1 reaction (µl) ABL: 14+1 reactions (µl) BCR-ABL Mbcr: 16+1 reactions (µl) Final concentration Freshly prepared LightCycler TaqMan Master Mix, 5x Primers and probe mix, 25x Nucleasefree PCR grade water Sample (to be added at step 4) Total volume 4.0 60 68.0 1x 0.8 12 13.6 1x 10.2 153 173.4 5.0 5 each 5.0 each 20.0 20 each 20.0 each 3. Dispense 15 µl of the qpcr pre-mix per capillary. 4. Add 5 µl of the RT product (cdna, 100 ng RNA equivalent) obtained in the reverse transcription (see Protocol: Recommended standardized EAC reverse transcription, page 14) in the corresponding tube (total volume 20 µl). 5. Mix gently, by pipetting up and down. 6. Place the capillaries in the adapters provided with the apparatus, and briefly centrifuge (700 x g, approximately 10 seconds). 7. Load the capillaries into the thermal cycler according to the manufacturer recommendations. 8. Program the LightCycler 1.2 or 2.0 Instrument with the thermal cycling program as indicated in Table 13. 28 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Table 13. Temperature profile Mode of analysis Quantification Hold Temperature: 95 C Time: 10 minutes Ramp: 20 Cycling 50 times 95 C for 10 seconds; ramp: 20 60 C for 1 minute; ramp: 20; with acquisition of FAM fluorescence: Single Hold 2 45 C for 1 minute; ramp: 20 9. For the LightCycler 1.2, follow step 9a. For the LightCycler 2.0, follow step 9b. 9a. LightCycler 1.2: The F1/F2 and 2 nd derivative analysis mode is recommended. Start the thermal cycling program, as indicated in Table 13. 9b. LightCycler 2.0: We recommend using Automated (F max) analysis on LightCycler 2.0 Software version 4.0 to obtain reproducible results. Start the thermal cycling program, as indicated in Table 13. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 29

Protocol: qpcr on the SmartCycler instrument Using this instrument, we recommend measuring samples in duplicate and controls only once, as indicated in Table 14. Table 14. Number of reactions for the SmartCycler instrument Samples Reactions With the ABL primers and probe mix (PPC-ABL) n cdna samples ABL standard Water control n x 2 reactions 1 x 3 reactions (3 standard dilutions, each one tested once) 1 reaction With the BCR-ABL Mbcr primers and probe mix (PPF-Mbcr) n cdna samples Mbcr standard Water control n x 2 reactions 1 x 5 reactions (5 standard dilutions, each one tested once) 1 reaction Sample processing on the SmartCycler instrument We recommend testing at least 5 cdna samples in the same experiment to optimize the use of the standards and primers and probe mixes. The two-block scheme in Figure 7 shows an example. Figure 7. Suggested plate setup for one experiment. S: cdna sample; F1 5: BCR-ABL Mbcr standards; C1 3: ABL standards; H 2 O: water control. qpcr on the SmartCycler instrument Note: Perform all steps on ice. 30 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Procedure 1. Thaw all necessary components and place them on ice. 2. Prepare the following qpcr mix according to the number of samples being processed. All concentrations are for the final volume of the reaction. Table 15 describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final reaction volume of 25 µl. A pre-mix can be prepared, according to the number of reactions, using the same primer and probe mix (either PPC-ABL or PPF-Mbcr). Extra volumes are included to compensate for pipetting error. Table 15. Preparation of qpcr mix Component TaqMan Universal PCR Master Mix, 2x Primers and probe mix, 25x Nucleasefree PCR grade water Sample (to be added at step 4) Total volume 1 reaction (µl) ABL: 14+1 reactions (µl) BCR-ABL Mbcr: 16+1 reactions (µl) Final concentration 12.5 187.5 212.5 1x 1 15 17 1x 6.5 97.5 110.5 5 5 each 5 each 25 25 each 25 each 3. Dispense 20 µl of the qpcr pre-mix per well. 4. Add 5 µl of the RT product (cdna, 100 ng RNA equivalent) obtained in the reverse transcription (see Protocol: Recommended standardized EAC reverse transcription, page 14) in the corresponding well (total volume 25 µl). ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 31

5. Mix gently, by pipetting up and down. 6. Load the samples into the thermal cycler according to the manufacturer recommendations. 7. Program the SmartCycler instrument with the thermal cycling program as indicated in Table 16. Table 16. Temperature profile Hold Temperature: 50 C Time: 2 minutes Hold 2 Temperature: 95 C Time: 10 minutes Cycling 50 times 95 C for 15 seconds 60 C for 1 minute with acquisition: Single 8. We recommend a threshold set at 30. Start the thermal cycling program, as indicated in Table 16. 32 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Interpretation of Results Data analysis principle Using TaqMan technology, the number of PCR cycles necessary to detect a signal above the threshold is called the threshold cycle (C T ) and is directly proportional to the amount of target present at the beginning of the reaction. Using standards with a known number of molecules, one can establish a standard curve and determine the precise amount of target present in the test sample. The ipsogen standard curves are plasmid-based; we use 3 plasmid standard dilutions for the CG, and 5 standard dilutions for the FG, in order to ensure accurate standard curves. Figures 8 and 9 show an example of TaqMan amplification curves obtained with the ipsogen BCR-ABL Mbcr Kit. Figure 8. Detection of BCR-ABL Mbcr standards (F1 F5). 10 1, 10 2, 10 3, 10 5, 10 6 copies/5 µl. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 33

Figure 9. Detection of ABL standards (C1, C2, C3). 10 3, 10 4, and 10 5 copies/5 µl. Results Standard curve and quality criteria Raw data can be pasted into an Excel file for analysis. For each gene (ABL and BCR-ABL), raw C T values obtained from plasmid standard dilutions are plotted according to the log copy number (3, 4, and 5 for C1, C2, and C3; 1, 2, 3, 5, and 6 for F1, F2, F3, F4, and F5). Figure 10 shows an example of the theoretical curve calculated on 5 standard dilutions. 34 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Figure 10. Theoretical curve calculated from the 5 standard dilutions. A linear regression curve (y = ax + b) is calculated for each gene (ABL and BCR-ABL), where a is the slope of the line and b is the y-intercept, which is the y-coordinate of the point where the line crosses the y axis. Its equation and coefficient of determination (R²) are printed on the graph. As standards are tenfold dilutions, the theoretical slope of the curve is 3.3. A slope between 3.0 and 3.9 is acceptable as long as R² is >0.95 (7). However, a value for R² >0.98 is desirable for precise results (3). Normalized copy number (NCN) The ABL standard curve equation should be used to transform raw C T values (obtained with PPC-ABL) for the unknown samples into ABL copy numbers (ABL CN ). The BCR-ABL standard curve equation should be used to transform raw C T values (obtained with PPF-Mbcr) for the unknown samples, into BCR-ABL copy numbers (BCR-ABL Mbcr CN ). The ratio of these CN values gives the normalized copy number (NCN): NCN = BCR-ABL Mbcr CN ABL CN x 100 MRD value The minimal residual disease (MRD) value is the ratio between the CG normalized expression of the FG in follow-up (FG CN /CG CN ) FUP and diagnostic samples (FG CN /CG CN ) DX. MRD value (MRDv) = (FG CN /CG CN ) FUP (FG CN /CG CN ) DX ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 35

Sensitivity The sensitivity (SENSv) is calculated according to the relative expression of the FG at diagnosis (FG CN /CG CN ) DX and CG expression (CG CN,FUP ) in the follow-up sample. Sensitivity (SENSv) = CG CN,DX CG CN,FUP x FG CN,DX Quality control on ABL values Poor quality of the RNA or problems during the qpcr steps result in low ABL CN. We recommend discarding results from samples giving ABL CN <4246.2 (lower value of the 95% CI from CML patient samples in the EAC study, reference 8). Reproducibility between replicates The variation in C T values between replicates should be <2, corresponding to a fourfold change in copy number values. Variation in C T values between replicates is generally <1.5 if the mean C T value of the replicates is <36 (7). Note: Each user should measure their own reproducibility in their laboratory. Water controls Negative controls should give zero CN. A positive water control results from a cross-contamination. See Troubleshooting guide, below, to find a solution. Troubleshooting guide This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical Support Center: www.qiagen.com/faq/faqlist.aspx. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocol in this handbook or sample and assay technologies (for contact information, see Contact Information, page 47). 36 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Comments and suggestions Negative result for the control gene (ABL) and BCR-ABL Mbcr in all the samples standard okay a) Poor RNA quality Always check the RNA quality and concentration before starting. Run a cell line RNA positive control (ipsogen BCR-ABL1 Mbcr Controls Kit, cat. no. 670191) in parallel. b) Failure of reverse transcription step Always check the RNA quality and concentration before starting. Run a cell line RNA positive control (ipsogen BCR-ABL1 Mbcr Controls Kit, cat. no. 670191) in parallel. Negative result for the control gene (ABL) in the samples standard okay a) Poor RNA quality Always check the RNA quality and concentration before starting. Run a cell line RNA positive control (ipsogen BCR-ABL1 Mbcr Controls Kit, cat. no. 670191) in parallel. b) Failure of reverse transcription step Always check the RNA quality and concentration before starting. Run a cell line RNA positive control (ipsogen BCR-ABL1 Mbcr Controls Kit, cat. no. 670191) in parallel. Standard signal negative a) Pipetting error Check pipetting scheme and the setup of the reaction. Repeat the PCR run. b) Inappropriate storage of kit components Store the ipsogen BCR-ABL1 Mbcr Kit at 15 to 30 C and keep primers and probe mixes (PPC and PPF) protected from light. See Reagent Storage and Handling, page 13. Avoid repeated freezing and thawing. Aliquot reagents for storage. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 37

Negative controls are positive Cross-contamination No signal, even in standard controls a) Pipetting error or omitted reagents Comments and suggestions Replace all critical reagents. Repeat the experiment with new aliquots of all reagents. Always handle samples, kit components, and consumables in accordance with commonly accepted practices to prevent carry-over contamination. Check pipetting scheme and the setup of the reaction. Repeat the PCR run. b) Inhibitory effects of the sample material, caused by insufficient purification c) LightCycler: Incorrect detection channel chosen d) LightCycler: No data acquisition programmed Repeat the RNA preparation. Set Channel Setting to F1/F2 or 530 nm/640 nm. Check the cycle programs. Select acquisition mode single at the end of each annealing segment of the PCR program. Absent or low signal in samples but standard controls okay a) Poor RNA quality or low concentration Always check the RNA quality and concentration before starting. Run a cell line RNA positive control (ipsogen BCR-ABL1 Mbcr Controls Kit, cat. no. 670191) in parallel. b) Failure of reverse transcription step Always check the RNA quality and concentration before starting. Run a cell line RNA positive control (ipsogen BCR-ABL1 Mbcr Controls Kit, cat. no. 670191) in parallel. 38 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Fluorescence intensity too low a) Inappropriate storage of kit components Comments and suggestions Store the ipsogen BCR-ABL1 Mbcr Kit at 15 to 30 C and keep primers and probe mixes (PPC and PPF) protected from light. See Reagent Storage and Handling, page 13. Avoid repeated freezing and thawing. Aliquot reagents for storage. b) Very low initial amount of target RNA Increase the amount of sample RNA. Note: Depending of the chosen method of RNA preparation, inhibitory effects may occur. LightCycler: Fluorescence intensity varies a) Pipetting error Variability caused by so-called pipetting error can be reduced by analyzing data in the F1/F2 or 530 nm/640 nm mode. b) Insufficient centrifugation of the capillaries c) Outer surface of the capillary tip dirty The prepared PCR mix may still be in the upper vessel of the capillary, or an air bubble could be trapped in the capillary tip. Always centrifuge capillaries loaded with the reaction mix as described in the specific operating manual of the apparatus. Always wear gloves when handling the capillaries. LightCycler: Error of the standard curve Pipetting error Variability caused by so-called pipetting error can be reduced by analyzing data in the F1/F2 or 530 nm/640 nm mode. Quality Control Quality control of the complete kit has been performed on a LightCycler 480 Instrument. This kit is manufactured according to ISO 13485:2003 standard. Certificates of analysis are available on request at www.qiagen.com/support/. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 39

Limitations The users must be trained and familiar with this technology prior to the use of this device. Any diagnostic results generated must be interpreted in conjunction with other clinical or laboratory findings. It is the user s responsibility to validate system performance for any procedures used in their laboratory which are not covered by the QIAGEN performance studies. Attention should be paid to expiration dates printed on the box and labels of all components. Do not use expired components. Note: The kit has been designed according to the Europe Against Cancer (EAC) studies (8), and is compliant with the updated international recommendations (3, 5). It should be used following the instructions given in this manual, in combination with validated reagents and instruments (see Materials Required but Not Provided, page 11). Any off-label use of this product and/or modification of the components will void QIAGEN s liability. Performance Characteristics Nonclinical studies Materials and methods Performance evaluation was performed on an ABI PRISM 7700 SDS, in combination with reagents listed in Materials Required but Not Provided, page 11. Equivalence studies validated its use on the following instruments: ABI PRISM 7000 and 7900HT SDS, LightCycler 1.2 and 480 Instruments, Rotor-Gene 3000, and SmartCycler instrument (9). Nonclinical studies were conducted to establish the analytical performance of the ipsogen BCR-ABL1 Mbcr Kit. These nonclinical laboratory studies were performed on total RNA from K562 cell line diluted in a constant final amount of MV4-11 cell line total RNA. To determine the repeatability of the assay, 5 different concentrations of K562 total RNA (5 ng, 500 pg, 50 pg, 5 pg, and 0.5 pg) diluted in MV4-11 total RNA, in a constant final total amount of 200 ng, were analyzed in 5 replicates per run and in 4 different runs (Figure 11). 40 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Figure 11. Amplification plots of 2.5 x 10 2 (5 ng), 2.5 x 10 4 (0.05 ng), 2.5 x 10 5 (0.005 ng), and 2.5 x 10 6 (0.0005 ng) dilutions of K562 total RNA in MV4-11 negative total RNA. Analytical data Tables 17 20 show the inter-assay analyses with the mean threshold cycle (C T ), standard deviation (SD), number of samples (n), coefficient of variation (CV), mean copy number (CN), and mean normalized copy number (NCN). Table 17. Inter-assay analysis cell lines BCR-ABL Mbcr and ABL Cell line BCR-ABL Mbcr Dilution Mean C T SD n CV (%) 2.5 x 10 2 (5 ng/200 ng) 26.18 0.40 20 1.54 2.5 x 10 3 (0.5 ng/200 ng) 29.32 0.53 19 1.82 2.5 x 10 4 (0.05 ng/200 ng) 32.62 0.62 20 1.91 ABL 23.59 0.20 95 0.83 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 41

Table 18. Inter-assay analysis plasmids Gene Plasmid Mean C T SD n CV (%) F1 (10 1 copies) 34.47 1.25 8 3.64 BCR-ABL Mbcr F2 (10 2 copies) 31.48 0.54 8 1.71 F3 (10 3 copies) 28.17 1.11 7 3.95 F4 (10 5 copies) 21.20 0.65 8 3.06 F5 (10 6 copies) 18.22 0.09 6 0.49 C1 (10 3 copies) 28.47 0.34 8 1.18 ABL C2 (10 4 copies) 25.25 0.31 8 1.22 C3 (10 5 copies) 21.92 0.70 8 3.19 Table 19. Inter-assay analysis cell lines BCR-ABL Mbcr and ABL (mean CN) Cell line Dilution Mean CN SD n CV (%) BCR-ABL Mbcr 2.5 x 10 2 (5 ng/200 ng) 2.5 x 10 3 (0.5 ng/200 ng) 2.5 x 10 4 (0.05 ng/200 ng) 4134.27 2512.40 20 60.77 512.8 479.51 19 93.51 42.94 22.05 20 51.36 ABL 33,831.51 13,637.7 94 40.31 Table 20. Inter-assay analysis cell line BCR-ABL Mbcr (mean NCN) Cell line Dilution Mean NCN* SD n CV (%) BCR- ABL Mbcr 2.5 x 10 2 (5 ng/200 ng) 12.6338 532.79 20 42.17 2.5 x 10 3 (0.5 ng/200 ng) 1.1605 94.69 19 81.61 2.5 x 10 4 (0.05 ng/200 ng) 0.1782 10.73 20 60.23 * For these study results only, the NCN is given as Mbcr CN ABL CN x 100. 42 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Clinical studies Performance evaluation was performed on an ABI PRISM 7700 SDS, in combination with reagents listed in Materials Required but Not Provided, page 11. Equivalence studies validated its use on the following instruments: ABI PRISM 7000 and 7900HT SDS, LightCycler 1.2 and 480 Instruments, Rotor-Gene 3000, and SmartCycler instrument (9). A group of 26 laboratories, in 10 European countries, organized in a Europe Against Cancer (EAC) concerted action, used plasmids provided by IPSOGEN to establish a standardized protocol for qpcr analysis of the major leukemiaassociated fusion genes in the clinical setting. The BCR-ABL p210 transcript was one of the fusion genes (FG) included in the study. We present here a summary of this validation study; full results have been published (8, 10). Inter-laboratory reproducibility for CG and FG plasmid standards Eleven laboratories performed an inter-laboratory reproducibility experiment to assess variability in the measurement of CG and FG plasmid standard dilutions. Dilutions were performed in duplicate at each facility. Table 21 reports the mean, standard deviation, and CV (%) for each dilution. Table 21. Inter-laboratory reproducibility for CG and FG plasmid standards Gene Dilution Mean C T SD CV (%) C1 29.59 1.34 4.54 ABL control gene C2 26.33 1.02 3.90 C3 22.75 1.59 6.97 F1 41.11 2.26 5.50 F2 37.43 1.51 4.04 BCR-ABL p210 fusion gene F3 33.76 1.28 3.81 F4 26.50 1.03 3.90 F5 22.98 0.97 4.21 Expression values of the BCR-ABL Mbcr FG transcript Tables 22 and 23 show the expression values of the BCR-ABL Mbcr FG transcript and ABL CG, for K562 cell line, CML and ALL patients at diagnosis, and normal patients. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 43

Table 22. Expression values of the BCR-ABL Mbcr FG transcript and ABL CG C T values BCR-ABL Mbcr C T values (95% range) ABL K562 cell line 20.5 20.7 CML patient samples BM (n = 15) 25.1 (21.5 27.0) 25.2 (20.7 26.8) PB (n = 14) 23.1 (21.9 25.8) 23.7 (22.6 26.7) ALL patient samples BM and PB (n = 17) 24.1 (21.5 29.9) 24.0 (21.6 26.4) Negative patient samples BM (n = 26) 25.35 (24.68 26.02) PB (n = 74) 25.15 (24.83 25.48) Table 23. Expression values of the BCR-ABL Mbcr FG transcript and ABL CG CN and ratio values CN values (95% range) BCR-ABL Mbcr ABL CML patient samples Ratio values (95% range)* CN BCR-ABL Mbcr/CN ABL BM (n = 15) 8710 (2089 112,202) 10,115.8 (4786.3 37,153.52) 0.86 (0.44 3.02) PB (n = 14) 17,783 (2042 112,202) 15,237 (4246.2 25,568.3) 1.17 (0.48 4.41) Negative patient samples BM (n = 26) PB (n = 74) 19,201 (12,922 25,480) 21,136 (17,834 24,437) * Results are expressed as simple BCR-ABL/ABL ratios. 44 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

ABL C T values did not differ significantly between normal and leukemia samples, nor between samples types (PB or BM) or leukemia samples (ALL, AML, CML). False positive and false negative rates False negative and false positive rates were computed using the following controls. Positive controls: K562 cells, a cell line well known for its positivity for BCR-ABL p210 fusion gene; patients samples already assessed for p210 positivity Negative controls: Negative RNA samples, no amplification controls (NAC) made of E. coli RNA instead of human RNA to check for PCR contamination, and no template controls (NTC), which contained water instead of human RNA Amplification on RNA samples of the FG was done in triplicate and in duplicate for the CG. A false-negative sample was defined as a positive RNA sample with less than 50% of positive wells (0/2, 0/3, or 1/3). A false-positive sample was defined as a negative sample with at least 50% of positive wells (1/2, 2/3, or 3/3). Table 24 shows the number and percentage of false negative and false positive samples. Table 24. False negative and false positive samples False negativity False positivity 10 3 10 4 FG negative control NAC/NTC 0% (0/33) 6.1% (2/33) 10.9% (6/55) 4.1% (14/340) References QIAGEN maintains a large, up-to-date online database of scientific publications utilizing QIAGEN products. Comprehensive search options allow you to find the articles you need, either by a simple keyword search or by specifying the application, research area, title, etc. For a complete list of references, visit the QIAGEN Reference Database online at www.qiagen.com/refdb/search.asp or contact QIAGEN Technical Services or your local distributor. ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 45

Cited references 1. Baccarani, M. et al. (2006) Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood 108, 1809. 2. Baccarani, M. et al. (2009) Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet. J. Clin. Oncol. 27, 6041. 3. Branford, S. et al. (2006) Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia. Leukemia 20, 1925. 4. Branford, S. et al. (2008) Desirable performance characteristics for BCR- ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials. Blood 112, 3330. 5. Hughes, T. et al. (2006) Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 108, 28. 6. White, H.E. et al. (2010) Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mrna. Blood 116, e111. 7. van der Velden, V.H., Hochhaus, A., Cazzaniga, G., Szczepanski, T., Gabert, J., and van Dongen, J.J. (2003) Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 17, 1013. 8. Gabert, J. et al. (2003) Standardization and quality control studies of real-time quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia a Europe Against Cancer program. Leukemia 17, 2318. 9. Silvy, M., Mancini, J., Thirion, X., Sigaux, F., and Gabert, J. (2005) Evaluation of real-time quantitative PCR machines for the monitoring of fusion gene transcripts using the Europe against cancer protocol. Leukemia 19, 305. 10. Beillard, E. et al. (2003) Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using realtime quantitative reverse-transcriptase polymerase chain reaction (RQ- PCR) - a Europe against cancer program. Leukemia 17, 2474. 46 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

Symbols The following symbols may appear on the packaging and labeling: <N> Contains reagents sufficient for <N> reactions Use by In vitro diagnostic medical device Catalog number Lot number Material number Global Trade Item Number Temperature limitation Manufacturer Consult instructions for use Contact Information For technical assistance and more information, please see our Technical Support Center at www.qiagen.com/support, call 00800-22-44-6000, or contact one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit www.qiagen.com). ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015 47

Ordering Information Product Contents Cat. no. ipsogen BCR-ABL1 Mbcr Kit (24) For 24 reactions: ABL Control Gene Standards, BCR-ABL Mbcr Fusion Gene Standards, Primer and Probe Mix ABL, Primer and Probe Mix BCR-ABL Mbcr Fusion Gene 670123 Rotor-Gene Q MDx for IVD-validated real-time PCR analysis in clinical applications Rotor-Gene Q MDx 5plex HRM Platform Real-time PCR cycler and High Resolution Melt analyzer with 5 channels (green, yellow, orange, red, crimson) plus HRM channel, laptop computer, software, accessories, 1-year warranty on parts and labor, installation and training not included 9002032 Rotor-Gene Q MDx 5plex HRM System Real-time PCR cycler and High Resolution Melt analyzer with 5 channels (green, yellow, orange, red, crimson) plus HRM channel, laptop computer, software, accessories, 1-year warranty on parts and labor, installation and training 9002033 ipsogen BCR-ABL1 Mbcr Controls Kit for qualitative validation of RNA extraction and reverse transcription of the BCR-ABL Mbcr fusion gene ipsogen BCR-ABL1 Mbcr Controls Kit Cell lines with negative, high, and low positive expression of the BCR-ABL Mbcr fusion gene 670191 For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. 48 ipsogen BCR-ABL1 Mbcr Kit Handbook 03/2015

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This product is intended for in vitro diagnostic use. ipsogen products may not be resold, modified for resale or used to manufacture commercial products without written approval of QIAGEN. Information in this document is subject to change without notice. QIAGEN assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall QIAGEN be liable for incidental, special, multiple, or consequential damages in connection with, or arising from the use of this document. ipsogen products are warranted to meet their stated specifications. QIAGEN s sole obligation and the customer's sole remedy are limited to replacement of products free of charge in the event products fail to perform as warranted. Trademarks: QIAGEN, ipsogen, Rotor-Gene (QIAGEN Group); ABI PRISM, FAM, RNaseOUT, SuperScript, SYBR, TAMRA (Life Technologies Corporation); Agilent, Bioanalyzer (Agilent Technologies, Inc.); Excel (Microsoft Corporation); LightCycler, TaqMan (Roche Group); SmartCycler (Cepheid). Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the ipsogen BCR-ABL1 Mbcr Kit to the following terms: 1. The ipsogen BCR-ABL1 Mbcr Kit may be used solely in accordance with the ipsogen BCR-ABL1 Mbcr Kit Handbook and for use with components contained in the Kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the ipsogen BCR-ABL1 Mbcr Kit Handbook and additional protocols available at www.qiagen.com. 2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties. 3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold. 4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated. 5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and/or its components. For updated license terms, see www.qiagen.com. HB-1360-002 2013 2015 QIAGEN, all rights reserved.

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